This study was designed to characterize muscarinic acetylcholine receptors (mAChRs) in primary cultured Sertoli cells from 30-d-old rats. RT-PCR was performed, and five PCR products corresponding to m1-m5 mAChR mRNA subtypes were detected in these cells. Ribonuclease protection assay further confirmed the presence of protected products for m1, m2, m3, and m4 mAChR transcripts. Radioligand binding studies and the analysis of changes in intracellular signaling pathways after cell exposure to carbachol were performed to study mAChRs at the protein level. Scatchard analysis revealed one single class of [H-3]quinuclidinyl benzilate binding sites. Carbachol produced a reduction on forskolin-induced intracellular cAMP accumulation in Sertoli cells. This effect was reversed by atropine, methoctramine, and tropicamide but not by p-fluoro-hexahydro-sila-difenidol or pirenzepine. Carbachol also induced an increase on total [H-3]-inositol phosphates content, an effect antagonized by atropine, p-fluoro-hexahydro-siladifenidol, or pirenzepine but not by methoctramine. Thus, mAChR activation in Sertoli cell is linked to both adenylyl cyclase inhibition and to phosphoinositide hydrolysis. Furthermore, gel shift assays indicated that carbachol also induced a time-dependent stimulation of the activator protein-1 DNA-binding activity, suggesting that activation of mAChRs may play a role in the modulation of gene expression in Sertoli cells. Taken together, these results indicate that mAChRs are present at mRNA and protein level in rat Sertoli cells.